The objectives of this ongoing project include the elucidation of (a) the mechanism of uptake of vitamin B6 by different organisms, (b) the interaction of its coenzyme forms, pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP), with various apoenzymes to form the corresponding holoenzymes and the effect of structural modification of both coenzyme and apoenzyme on this interaction, and (c) the catalytic mechanism of action of PLP-enzymes. To this end we have isolated several different bacterial PLP-enzymes in homogeneous form and are conducting extensive comparisons of their spectral and catalytic properties in the presence and absence of substrates and substrate analogues, and of the conditions necessary for their resolution from and reconstitution with PLP (e.g., how these processes are affected by pH, ionic strength, carbonyl reagents, protein modification, other proteins, etc.). We are also investigating the effects of modification of protein structure on catalytic properties of these enzymes: such modification can be affected either by chemical modification in structure of the coenyme, or by mutational alteration of the structure of the apoenzyme. In the latter case, the chemical basis for observed differences will be studied by defining whether the effect is on ability to retain the coenzyme, or upon catalytic activity per se; in either case attempts to define the effects of the mutational alteration in terms of amino acid substitutions will be made. Finally we will continue the use of isotopically labeled pyridoxine, pyridoxal, and pyridoxamine to study active transport of vitamin B6 and to determine to what extent differences in transport specificities determine activity of these compounds in promoting growth of auxotrophic organisms. Pyridoxine dehydrogenase will be isolated and studied to determine what its role in vitamin B6 metabolism is. Finally, the several possible phosphate esters of vitamin B6 will be synthesized and their behavior toward hydrolysis compared.